融合DNA疫苗CRT180/HPV6E7的构建及其免疫学特性研究

The Construction and Immune Response of Fusion DNA Vaccine CRT180/HPV6E7

目的构建融合DNA疫苗CRT180/HPV6E7,并评价其体外实验和动物模型上的免疫学特性,尤其是抗病毒致病基因的特异性细胞免疫反应。方法分别构建DNA重组体pcDNA3.1-CRT180/HPV6E7,pcDNA3.1-CRT180,pcDNA3.1-HPV6E7。将pcDNA3.1-HPV6E7质粒经脂质体介导转染B16细胞,以G418筛选阳性细胞集落,荧光显微镜观察和RT-PCR鉴定表达HPV6E7的阳性细胞株。将质粒DNA肌注免疫C57BL/6小鼠,3次后取全血经流式细胞仪检测T细胞亚群的变化,LDH法检测小鼠的脾细胞和淋巴细胞与靶细胞B16/HPV6E7孵育后的CTL活性以及ELISA法检测共孵育上清中IL-2和IFN-γ浓度的变化。结果各组质粒经鉴定表明构建正确。转染后细胞株稳定表达HPV6E7。DNA疫苗免疫小鼠后,pcDNA3.1-CRT180/HPV6E7免疫组较pcDNA3.1-HPV6E7免疫组的外周血CD8+T细胞和TCRγδT细胞的比例显著增加,脾细胞和淋巴细胞针对靶细胞B16/HPV6E7的CTL杀伤活性更明显,分泌IL-2和IFN-γ的水平升高(P<0.05)。结论CRT180与HPV致病基因6E7相连的重组质粒疫苗pcDNA3.1-CRT180/HPV6E7能引发小鼠T细胞亚群CD8+分化并诱导出显著的特异性CTL反应。推测CRT180/HPV6E7DNA疫苗在动物模型上可以有效激活抗HPV特异性细胞免疫,有利于病毒感染的清除。

Objective To investigate the immune response, in particular, the anti-virus cellular immune response induced by the constructed fusion DNA vaccine CRT （Calreticulin）180/HPV6E7 in vivo and vitro. Methods The HPV6E7 open reading frame was amplified by polymerase chain reaction from pUC/HPV6 plasmid. The CRT180 gene was cloned by reverse transcription from human muscle tissues. The PCR products of CRT180 and HPV6E7 were subcloned into pcDNA3. 1-GFP eukaryotlc vector. The recombinant plasmids CRT180/ HPV6E7 was authenticated by restrict enzyme digestion and confirmed by DNA sequencing. The DNA plasmid HPV6E7 with report gene GFP was transfected to murine B16 cells by lipofectamine kit to establish the target cells. The HPV6ET-expressing cell line was selected by G418 and identified by RT-PCR. The C57BL/6 mice were vaccinated via intramuscular injection in the right legs with 100 μg plasmid encoding CRT180-HPV6E7, CRT180 or HPV6E7, empty plasmid without insert, and PBS group respectively. The changes of the T lymphocyte subsets in the peripheral blood of the mice were evaluated by flow cytometric analysis. The lymphocytes from the spleen and lymph nodes were harvested and co-cultured with HPV6E7-expressing cell lines. The CTLs kill activity and the cytokines IL-2, IFN-Tsecretion levels of the lymphocytes were assessed by LDH and ELISA respectively. Results The constructed recombinant plasmids pcDNA3. 1-CRT180/HPV6ET, pcDNA3. 1-CRT180 and pcDNA3.1- HPV6E7 were authenticated by restrict enzyme digestion and confirmed by DNA sequencing. Green fluorescence located in the cellular nucleus and plasma could be detected by fluorescent microscope after transfection with plasmids. The results of RT-PCR from the selected positive cell line showed the expected fragments of HPV6E7 mRNA. After immunization, the percentage of CD8^＋ or TCRγδT cells in the peripheral blood, the CTLs kill activity of the spleen ceils and lymphocytes against HPV6E7-expressing cells, and the secretion levels of IL-2,IFN-7 in CRT180/HPV6E7 vaccinated group increased significantly compared with the control groups （P〈0. 05）. Conclusion Vaccination with fusion DNA vaccine CRT180/HPV6E7 could elicit a more efficient HPV6E7-specific immune response than with HPV6E7 plasmid, indicating the potential of CRT180/ HPV6E7 vaccine to enhance the antigen presentation. The recombinant CRT180/HPV6E7 might help the elimination of virus in animal models and accordingly be used as a vaccine candidate in the therapy of CA.